Method for isomerizing double bonds

ABSTRACT

A method for isomerizing a double bond is provided. A substrate is exposed to an isomerase enzyme, wherein the isomerase enzyme comprises a redox-regulated ligand switch and heme b cofactor. A reducing agent is added which changes iron (III) to iron (II). The enzyme isomerizes double bonds in the iron (II) state but not in the iron (III) state. In one embodiment, the enzyme is homologous with 15-cis-ζ-carotene isomerase (Z-ISO).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a non-provisional of U.S.Patent Application Ser. No. 62/168,994 (filed Jun. 1, 2015), theentirety of which is incorporated herein by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contract numberGM081160 awarded by the National Institute of Health (NIH). Thegovernment has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

This application refers to a “Sequence Listing” listed below, which isprovided as an electronic document entitled “Sequence_ST25.txt” (10 kb,created on May 18, 2016), which is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

The subject matter disclosed herein relates to methods for isomerizingdouble bonds using chemical catalysts. Plants synthesize carotenoids,which are essential metabolites for plant development and survival.These metabolites also serve as essential nutrients for human health.The biosynthetic pathway for all plant carotenoids occurs inchloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase(Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone orin combination with other enzymes.

Carotenoids constitute a large class of isoprenoids synthesized by allphotosynthetic organisms, some bacteria, fungi and arthropods. Globalvitamin A deficiency in children has sparked worldwide efforts toincrease the levels of provitamin A carotenoids in food-crop staples.This goal rests on furthering knowledge of how plants control andbiosynthesize carotenoids that can be converted in humans to vitamin A.Metabolic engineering and breeding of plants rich in particularcarotenoids will continue to be an important objective for addressingthe challenges of providing food security in a changing climate.Carotenoid functions are central to plant growth and development. Theplant carotenoid-biosynthetic pathway is mediated by enzymes encoded inthe nucleus and localized to chloroplasts or other plastids. Thecarotenoid-biosynthetic reactions begin with formation of the colorless15-cis-phytoene, which undergoes desaturation and isomerization ofdouble bonds to create carotenoids with yellow, red and orange colors.The pathway requires an electron-transfer chain and plastoquinones tochannel electrons and protons produced during desaturation mediated byphytoene desaturase (PDS) and ζ-carotene desaturase (ZDS). PDS produces9,15,9′-tri-cis-ζ-carotene, which must be isomerized at the 15-15′-ciscarbon-carbon double bond to form 9,9′-di-cis-ζ-carotene, the substratefor a second desaturase, ZDS (FIG. 1A). Although light can partiallymediate this cis-trans carbon-carbon isomerization, Z-ISO is essential,especially in tissues receiving no light exposure, such as the endospermtissue, which has been targeted for improvement of provitamin Acarotenoids in efforts to alleviate global vitamin A deficiency. Plantswith insufficient Z-ISO also grow poorly under the stress of fluctuatingtemperature. Because climatic variations alter the need forphotosynthetic and nonphotosynthetic carotenoids, Z-ISO facilitatesplant adaptation to environmental stress, a major factor affecting cropyield. Thus, Z-ISO is essential for maximizing plant fitness in responseto environmental changes and for promoting accumulation of provitamin Acarotenoids in edible tissues.

Mutants blocked in Z-ISO function accumulate 9,15,9′-tri-cis-ζ-carotene,the putative Z-ISO substrate. When the gene encoding Z-ISO is introducedinto Escherichia coli cells producing 9,15,9′-tri-cis-ζ-carotene, thiscarotenoid is isomerized into the putative Z-ISO product,9,9′-di-cis-ζ-carotene. These data suggest that Z-ISO is required forisomerization of the 15-cis bond in 9,15,9′-tri-cis-ζ-carotene but notthe 15-cis bond in 15-cis-phytoene. In E. coli experiments, theisomerization activity associated with Z-ISO occurred in the presence ofseveral upstream carotenoid-biosynthetic enzymes needed to produce theZ-ISO substrate. It is therefore desirable to determine whether Z-ISO isa bona fide enzyme that catalyzes isomerization through a uniquemechanism requiring a redox-regulated heme cofactor.

The discussion above is merely provided for general backgroundinformation and is not intended to be used as an aid in determining thescope of the claimed subject matter.

BRIEF DESCRIPTION OF THE INVENTION

A method for isomerizing a double bond is provided. A substrate isexposed to an isomerase enzyme, wherein the isomerase enzyme comprises aredox-regulated ligand switch and heme b cofactor. A reducing agent isadded which changes iron (III) to iron (II). The enzyme isomerizesdouble bonds in the iron (II) state but not in the iron (III) state. Inone embodiment, the enzyme is homologous with 15-cis-ζ-caroteneisomerase (Z-ISO). An advantage that may be realized in the practice ofsome disclosed embodiments of the method is that an alternative catalystis provided that can isomerize double bonds.

In a first embodiment, a method for isomerizing a double bond isprovided. The method comprises exposing a substrate to an isomeraseenzyme, wherein the isomerase enzyme comprises a redox-regulated ligandswitch and heme b cofactor, wherein the heme b cofactor comprises aniron having an iron (III) oxidation state and the substrate comprises adouble bond in the substrate with a cis stereochemistry; exposing theisomerase enzyme to a reducing agent such that the isomerase enzymechanges from the iron (III) oxidation state to an iron (II) oxidationstate, wherein the isomerase enzyme isomerizes a double bond when in theiron (II) oxidation state and does not isomerize the double bond when inthe iron (III) oxidation state; and permitting the double bond in thesubstrate to undergo isomerization from the cis stereochemistry to atrans stereochemistry, wherein the isomerization is catalyzed by theisomerase enzyme.

In a second embodiment, a method for isomerizing a double bond isprovided. The method comprises sequential steps of exposing a carotenoidsubstrate to an isomerase enzyme, wherein the isomerase enzyme comprisesa redox-regulated ligand switch and heme b cofactor, wherein the heme bcofactor comprises an iron having an iron (III) oxidation state and thecarotenoid substrate comprises a double bond in the carotenoid substratewith a cis stereochemistry, wherein the isomerase enzyme has fewer thanfour hundred residues and comprises SEQ ID NO. 11; exposing theisomerase enzyme to a reducing agent in vitro such that the isomeraseenzyme changes from the iron (III) oxidation state to an iron (II)oxidation state, wherein the isomerase enzyme isomerizes a double bondwhen in the iron (II) oxidation state and does not isomerize the doublebond when in the iron (III) oxidation state; and permitting a doublebond in the carotenoid substrate to undergo isomerization from the cisstereochemistry to a trans stereochemistry, wherein the isomerization iscatalyzed by the isomerase enzyme.

This brief description of the invention is intended only to provide abrief overview of subject matter disclosed herein according to one ormore illustrative embodiments, and does not serve as a guide tointerpreting the claims or to define or limit the scope of theinvention, which is defined only by the appended claims. This briefdescription is provided to introduce an illustrative selection ofconcepts in a simplified form that are further described below in thedetailed description. This brief description is not intended to identifykey features or essential features of the claimed subject matter, nor isit intended to be used as an aid in determining the scope of the claimedsubject matter. The claimed subject matter is not limited toimplementations that solve any or all disadvantages noted in thebackground.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the features of the invention can beunderstood, a detailed description of the invention may be had byreference to certain embodiments, some of which are illustrated in theaccompanying drawings. It is to be noted, however, that the drawingsillustrate only certain embodiments of this invention and are thereforenot to be considered limiting of its scope, for the scope of theinvention encompasses other equally effective embodiments. The drawingsare not necessarily to scale, emphasis generally being placed uponillustrating the features of certain embodiments of the invention. Inthe drawings, like numerals are used to indicate like parts throughoutthe various views. Thus, for further understanding of the invention,reference can be made to the following detailed description, read inconnection with the drawings in which:

FIG. 1A depicts a cis-trans isomerization of 15-cis-ζ-carotene isomerase(Z-ISO) to trans Z-ISO;

FIG. 1B is graph depicting the conversion of cis Z-ISO to trans Z-ISO isonly catalyzed in the presence of a reduced enzyme complex;

FIG. 2A is a schematic depiction of Z-ISO in an oxidized (inactive)state and in a reduced (active) state;

FIG. 2B is a UV-vis absorption spectrum from a pyridine hemochrome assayof nickel affinity-purified MBP::Z-ISO protein extract;

FIG. 2C is a UV-vis absorption spectrum from MBP::Z-ISO in isolated(oxidized), dithionite reduced or dithionite reduced and CO treatedstates;

FIG. 2D is a difference spectrum of data from FIG. 2C;

FIG. 2E is a UV-vis absorption spectrum showing binding of cyanide (2mM) to as-purified Z-ISO (21 μM) wherein the inset is the differencespectra comparing cyanide binding to dithionite-reduced enzyme);

FIG. 3A is an EPR spectrum of as-purified Z-ISO;

FIG. 3B is an EPR spectrum showing an enlarged view of the low-spinregion of FIG. 3A;

FIG. 4A depicts MCD and UV-vis spectra of as-purified Fe(III) Z-ISO(oxidized) compared with spectra of a 50-50 mixture of Fe(III)cytochrome (cyt)b₅ (bis-histidine) and imidazole (Im)-bound Fe(III)P450_(cam) (histidine-cysteine);

FIG. 4B depicts MCD and UV-vis spectra of as-dithionite-reduced ferrous(Fe(II)) Z-ISO compared with spectra of mono and bis Im-bound H93G Mb;

FIG. 5A is a graph depicting isomerization activity, tested byfunctional complementation in E. coli, with carotenoid products measuredby HPLC;

FIG. 5B and FIG. 5C are UV-vis spectra normalized for absorbance at 280nm for extracted mutant fusion proteins, which were as purified(oxidized) or dithionite reduced;

FIG. 6A is a Z-ISO homology model illustrating how proximity ofalternate heme ligands predicts feasibility of distal-ligand switchingbetween H266 and C263 when heme proximal ligand is H150; and

FIG. 6B is a schematic of heme-ligand states based on experimentalevidence;

DETAILED DESCRIPTION OF THE INVENTION

Without wishing to be bound to any particular theory, carotenoidisomerization of the central double bond is believed to involve15-cis-ζ-carotene isomerase (Z-ISO) with an unusual heme-dependentchemistry. See FIG. 2A. Z-ISO has been identified as a bona fide enzymeand integral membrane protein. Z-ISO independently catalyzes thecis-trans isomerization of the 15-15′ carbon-carbon double bond in9,15,9′-cis-ζ-carotene to produce the substrate required by thesubsequent biosynthetic-pathway enzyme. This isomerization depends upona ferrous heme b cofactor that undergoes redox-regulated ligandswitching between the heme iron and alternate Z-ISO amino acid residues.Heme b-dependent isomerization of a large hydrophobic compound in amembrane was previously undescribed. As an isomerase, Z—ISO represents anew prototype for heme b proteins and potentially uses a new chemicalmechanism.

Two isoforms of Z-ISO are provided in SEQ ID NO. 9 and SEQ ID NO. 10.SEQ ID NO. 10 is the same as SEQ ID NO. 9 except in that residues257-285 are different and residues 286-367 are omitted. Both isoformsshare a common sequence given by SEQ ID NO. 11. In one embodiment, anisomerase enzyme is used than has fewer than four hundred residues andcomprises the common sequence given by SEQ ID NO. 11. In anotherembodiment, the isomerase enzyme is SEQ ID NO. 9 or SEQ ID NO. 10. Inanother embodiment, the isomerase enzyme is homologous with (e.g. atleast 70% homologous, 80% homologous, 90% homologous or 95% homologous)either SEQ ID NO. 9 or SEQ ID NO. 10

Z-ISO and its related protein sequences, including the NnrU protein,contain a redox-regulated ligand switch and heme b cofactor which can beused to control the cis to trans isomerization of C═C and other doublebonds, including N═O, and for control of carotenoid biosynthesis fluxand carotenoid levels through Z-ISO heme redox state and throughmanipulation of internal ligands and external ligands, including bindingof substrates and diatomic gasses to the heme iron. The enzyme is in aninactive state when the iron is in a +3 oxidation state. The enzyme isin an active state when the iron is in a +2 oxidation state. Theactivity of the enzyme can therefore be controlled by controlling theredox conditions of the enzyme environment.

This disclosure provides knowledge of the Z-ISO mechanism that will nowallow for post-translational control of the biosynthesis of carotenoids,as well as the control of the cis to trans isomerization of double bondscontained by other synthetic (non-native) substrates.

In other embodiments, by modifying the Z-ISO (and other relatedsequences including NnrU) protein sequence from any organism, includingmodification of the heme ligand residues, will result in creation of newenzymes that recognize novel substrates on which Z-ISO can catalyze thecis to trans isomerization of double bonds to form compounds with novelchemical properties. After benefitting from reading this specification,those skilled in the art will appreciate such new enzymes can begenerated for specific substrates using conventional biotechnologytechniques including, for example, mutagenesis.

To directly test whether Z-ISO is a bona fide enzyme an in vitro assaywas developed with isolated Z-ISO from Zea mays and artificial liposomescontaining the Z-ISO substrate. First, the substrate from E. coli waspurified and combined with lipids to form artificial liposomes. Next,Z-ISO was overexpressed and purified as a TEV protease-cleavablemaltose-binding protein (MBP) fusion (MBP::Z-ISO). Last, the isolatedfusion protein of 90% purity was incubated with TEV protease to cleaveZ-ISO away from the fused MBP before initiation of the isomerizationreaction. Conversion of the substrate to product occurred only in thepresence of Z-ISO that had been pretreated with dithionite to a finalconcentration of 10 mM to create reducing conditions (FIG. 1B). Theas-purified enzyme (considered to be oxidized), as well asheat-denatured Z-ISO, was not functional. The Z-ISO catalyzedisomerization only when the reaction was conducted under reducingconditions but not oxidizing conditions. In this experiment, theliposomes used for the in vitro assay were also essential, becausereactions lacking liposomes also did not work.

To gain insight into the mechanism of isomerization catalytic motifs orother characteristic domains in Z-ISO were identified. BLAST analysissuggested that, although Z-ISO is highly conserved in plants, it sharessequence homology (about 76% similarity) with only NnrU, anuncharacterized membrane protein associated with nitric oxide (NO)metabolism in noncarotenogenic bacteria that perform denitrification. Inaddition, a chloroplast-targeting sequence was previously identified inZ-ISO, thus suggesting that Z-ISO is a plastid-localized protein.Bioinformatic approaches were used to generate hypotheses on thelocation and function of Z-ISO and tested them further.

MEMSAT3 predicted seven transmembrane (TM) domains in maize Z-ISO, withTM2-TM7 showing homology to the corresponding TM domains in NnrU. Incontrast to a functional Arabidopsis transcript (ZISOJ1.1), a shorterArabidopsis transcript (ZISO1.2) encodes a nonfunctional protein withone less TM domain at the C terminus. The effect of the deletionsuggests that the C-terminal M domain is important for the function (forexample, activity or proper folding) of Z-ISO.

To test the prediction that Z-ISO is targeted to the chloroplast, thegene encoding GFP was fused downstream of the gene encoding Z-ISO,including its transit peptide. The fusion construct was transientlyexpressed in maize leaf protoplasts. GFP fluorescence confirmed thatZ-ISO colocalized in the chloroplasts together with chlorophyll). Invitro chloroplast protein import demonstrated that Z-ISO is achloroplast integral membrane protein, as predicted by the topologypredictions. When taken together, these observations suggest that Z-ISOis localized in chloroplast membranes. Z-ISO was also found to exist ina high-molecular-weight protein complex of about 480 kDa, as similarlynoted for other carotenoid enzymes.

Next, homology-modeling tools were applied to look for structuralhomologies missed by the BLAST analysis. Homology modeling was expectedto be limited by the underrepresentation of membrane-protein structuresin the Protein Data Bank, owing to the inherent difficulties incrystallizing membrane proteins. Homology modeling of Z-ISO with theMeta Server program modeled the residues of Z-ISO onto an integralmembrane protein, the diheme cytochrome b subunit of quinol-fumarateoxidoreductase. The fold-recognition program LOOPP predicted that Z-ISOmight contain nonheme iron. These programs are based on uniquealgorithms, and therefore the templates chosen for modeling by theprograms were different. However, neither NnrU nor Z-ISO had beenannotated as metalloproteins.

To test the prediction that Z-ISO is a metalloenzyrne, inductivelycoupled plasma optical emission spectrometry (ICP-OES) was used tomeasure the metal content. The result showed that iron, but not calcium,copper, nickel, magnesium, manganese, molybdenum or zinc, is present inthe MBP::Z-ISO fusion. Because MBP is not a metalloprotein, theprotein-bound iron was postulated to be exclusively associated withZ-ISO. Cultures with MBP::Z-ISO are brown, as is the purified protein,consistent with the presence of heme or nonheme iron.

To test specifically for heme, MBP::Z-ISO was separated and Z-ISO andMBP were cleaved by SDS-PAGE and stained for heme and then for totalprotein. The results showed that both MBP::Z-ISO and Z-ISO containedheme, whereas MBP did not. A pyridine hemochrome assay was conducted(FIG. 2B) to examine the heme cofactor independent of Z-ISO and foundthat it is a heme b, on the basis of spectroscopic signature. Therelated NnrU protein is also known to contain a heme b. UV-visible(UV-vis) absorption spectroscopy of as-purified Z-ISO together with itsbound heme indicated the presence of an oxidized, ferric (Fe(III) state)heme. To generate the spectrum of the reduced Z-ISO heme (with a ferrous(Fe(II)) heme iron, the as-purified Z-ISO was treated with dithionite.The spectrum of the dithionite-reduced Z-ISO (FIG. 2C) is similar tospectra of cytochromes containing heme b with two axial histidineligands. Carbon monoxide (CO) was used as a diagnostic probe to testwhether the heme could coordinate electrons with an exogenous ligand.The shift in the UV-vis spectrum attributed to the heme indicated thatCO could bind and coordinate to the heme iron of Z-ISO (FIG. 2C and FIG.2D). The binding was stoichiometric, given that the absorbanceassociated with CO binding was almost equivalent to that associated withthe reduced heme. That is, the absorbance of the α-band trough at 560 nmin the CO difference spectrum (FIG. 2D) was about 90% the intensity ofthe α-band peak for the same sample in the reduced minus oxidizedspectrum. The comparison indicated that at least 90% of the reduced hemehad bound CO. This result suggests that one of the axial amino acidligands may be displaced by an exogenous ligand. The import of thisobservation is that the Z-ISO heme iron may not be limited to shuttlingelectrons, as in the case of hemes that participate in electrontransfer, but instead the Z-ISO heme iron may have a role in catalysis.

UV-vis absorption spectroscopy analysis showed that an exogenous ligandcan bind to the heme iron in the Fe(II) state, but it was not knownwhether an exogenous ligand could displace an axial ligand if the hemewere in the Fe(III) state. To test this possibility cyanide (CN⁻), whichis known to bind preferentially to ferric rather than ferrous heme, wasintroduced. Cyanide was added to both the as-purified enzyme withoxidized, Fe(III) heme and to the dithionite-reduced enzyme carrying areduced, Fe(II) heme and the UV-vis absorption was measured. See FIG.2E. Binding of cyanide to the Fe(III) heme of Z-ISO was observed undersaturating concentrations of KCN, as indicated by the shift in the Soretpeak (from 413 nm to 415 nm or 416 nm), and the new spectrum resembledthat of cyanomyoglobin, which has histidine as the protein-anchoring, orso-called proximal, ligand. However, binding of cyanide to the ferriciron was substoichiometric, on the basis of analysis of the differencespectra. Addition of cyanide- to the ferrous enzyme showed no spectralchange. These results support the presence of a pentacoordinate,high-spin monohistidine ligand-bound ferric heme (in equilibrium withlow-spin hexacoordinate heme), which can bind exogenous ligand in theoxidized, inactive enzyme. The substoichiometric binding of cyanidesuggests that this pentacoordinate, high-spin species represents a smallsubset of the total ferric heme.

An X-band EPR spectrum of the as-purified MBP::Z-ISO fusion proteinindicated the presence of a high-spin ferric heme (i.e., heme b with anaxial histidine ligand) at a proportionality factor (g): of 5.8 and ofmultiple low-spin hemes with broad EPR signals (FIG. 3A and FIG. 3B). Inaddition, a minor nonheme-iron species was observed at g of 4.3, whichwas postulated to be nonspecifically bound to Z-ISO. The low-spin hemeEPR signals (shown in FIG. 3A and FIG. 3B) are consistent with theexistence of two major types of low-spin heme species with either abis-histidine or histidine-cysteine axial ligand set, respectively. Thelow-spin species at g of 2.98 is assigned as a hexacoordinate heme witha bis-histidine axial ligand set, on the basis of the similarity of itsg factors to those of other heme species with a bis-imidazole axialligand set. The signals at g of 2.54, 2.50 and 2.43 are attributed tothe g_(x) tensors for multiple components of a hexacoordinate low-spinheme species with a histidine-cysteine axial ligand set. Such low-spinspecies typically display a narrow distribution of the g factors, owingto pronounced delocalization of the spin density to the cysteine ligand.The heterogeneity of this histidine-cysteine-coordinated heme speciesprobably originated from variations in the coordination position as wellas the protonation or hydrogen-bonding state of the cysteine ligand.Previous studies on other systems have demonstrated that the g factorsfor histidine-cysteine-coordinated heme species are sensitive to theelectronic properties of the heme environment and the protonation stateof the axial ligands. Next, the as-purified sample was chemicallyreduced with dithionite and the reduced sample was EPR silent. Withaddition of NO, a strong EPR signal at the g=2 region was detected,which was attributed to the formation of a low-spin hexacoordinateFe(II)-nitrosyl heme complex. NO binding is consistent with the findingthat reduced MBP::Z-ISO also binds CO (FIG. 2C and FIG. 2D). The EPRspectrum of the Fe(II)-nitrosyl complex of MBP::Z-ISO is similar tospectra of other Fe(II)-nitrosyl adducts of histidine-ligated hemes,such as those reported in cytochrome c oxidase, cytochrome c peroxidase,heme oxygenase, hemoglobin, myoglobin and horseradish peroxidase, thussuggesting that a histidine residue is retained as the axial ligand ofthe ferrous heme with NO is bound.

EPR analysis revealed high-spin and low-spin hemes. A high-spin heme canhave an easily observable EPR signal, even if it is a minor component.To further examine the hemes in the same sample as used for EPR,magnetic circular dichroism (MCD) was used, which detects mainly theheme chromophore (300-700 nm). To ascertain a detection limit of thepercentage of high-spin heme in a sample containing a mixture oflow-spin and high-spin heme, the MCD and UV-vis absorption spectra ofFe(III) cytochrome b5 (100% low-spin species) was compared to Fe(III) Mb(met-aqua-Mb): (about 100% high spin) in a series of low-spin/high-spinmixtures (95:5, 90:10, 80:20 and 50:50). This comparison showed thatFe(III) Z-ISO (FIG. 4A) contains less than 20% (probably less than 10%)high-spin heme at ambient temperature, presumably from equilibriumdissociation of an axial ligand from the low-spin heme.

MCD also showed that ferric Z-ISO has two ligand pairs(histidine-histidine and histidine-cysteine), consistent with the EPRresults. This was determined by comparing the as-purified Z-ISO spectrumwith that of a simulated mixture of cytochrome b5 (bis-histidine) andimidazole (Im)-bound P450_(cam) (histidine-cysteine) (FIG. 4A). The datashow a good fit to two ligand-coordination modes in low-spin ferricZ-ISO at about a 1:1 ratio. If there is only one heme center in theprotein, histidine and cysteine might occupy the distal side of the hemeas alternative ligands while the proximal side is ligated by a commonhistidine. The MCD spectrum of the dithionite-reduced Z-ISO. MCD showeda single heme species in the reduced Z-ISO coordinated by bis-histidine(FIG. 4B). Importantly, the amount of reduced histidine-histidine hemewas equivalent to the combined concentration of the histidine-histidineand histidine-cysteine heme seen in oxidized Z-ISO.

If two heme centers exist, there should be two separate proximalhistidines. To distinguish between these alternate hypotheses, anotherapproach was used to identify all histidines in Z-ISO that could serveas heme ligands. A search was conducted for the specific residues thatmight function as Z-ISO heme ligands. Available Z-ISO sequences werealigned and all evolutionarily conserved residues were identified thathave been reported to serve as heme ligands. These were mutagenized toalanine and tested for activity with E. coli complementation. Of allconserved histidines, only two (H150 and H266) were required foractivity (FIG. 5A). Mutants with alanine substitution at H150 (H1SOA) orH266 (H266A), as compared to wild-type Z-ISO, decreased the conversionof substrate to product. Loss of the isomerization activity was not dueto absence of expression, the possibility of which was ruled out with ananti-maize Z-ISO polyclonal antiserum. Loss of either residue alsodisrupted heme binding, as evidenced by the reduction in bound heme forMBP-fusion proteins carrying the alanine variants and by the UV-visspectral shift seen for both the as-purified (oxidized) ordithionite-reduced proteins (FIG. 5B and FIG. 5C).

On the basis of the mutagenesis results, the two-heme model for Z-ISOwas ruled out. Two hemes would have necessitated a total of at leastthree histidines (two proximal and at least one distal), but noadditional conserved histidines that were required for activity beyondH150 and H266. Predicted locations of H150 and H266 from the Z-ISOhomology model (FIG. 6A) are consistent with coordination of a commoncofactor. Therefore the data are consistent with the presence of asingle heme that undergoes a change in axial ligation when reduced (FIG.6B).

The ability of Z-ISO to bind exogenous ligands indicates theavailability of an axial coordination site on its heme and faciledissociation of one of two axial ligands. The most extensivedissociation takes place in the reduced, active form of the enzyme.Z-ISO activity is predicated on the heme ligands H150 and H266 and onthe heme iron being in the reduced state. Therefore, it is possible thatthe heme iron directly mediates isomerization by interacting with thesubstrate. An alternative hypothesis is that, as a result ofredox-dependent ligand switching, the switch to bis-histidine exposesC263, which becomes accessible to mediate catalysis. A precedent forfunction of a cysteine residue in catalysis, particularly in double-bondisomerization, has been seen for isopentenyl diphosphate isomerase, anonheme enzyme that catalyzes double-bond isomerization. The C263alternate heme ligand is the only cysteine in Z-ISO, and it isevolutionarily conserved in all Z-ISO sequences. The cysteine residueappears unlikely to function in protein dimerization, because the invitro reaction included the reducing agent dithiothreitol, which wouldeliminate dimerization mediated by cysteine sulfhydryl bridges. If C263is essential for catalysis, then mutagenesis to a nonredox activeresidue should inactivate the enzyme. Mutation to alanine had no effecton activity or expression when the E. coli complementation system wasused (FIG. 5A). Although the C263A MBP-fusion variant carries a reducedamount of heme equivalent to the H266A variant, the UV-vis spectrum ofC263A is similar to that of wild-type Z-ISO (FIG. 5B and FIG. 5C). Whentaken together, these results suggest that C263 is not catalytic butinstead has a role in heme binding and reversible heme ligation.

The heme is likely to function as the mechanistic cofactor according tothe observations that, with loss of either of the apparent histidineligands (H150 or H266), Z-ISO becomes inactive, the heme spectrum isaltered, and heme binding is decreased. EPR and MCD spectroscopytogether identify the axial ligands as bis-histidine orhistidine-cysteine. Notably, H266 and C263 are only three residuesapart. Therefore, these two residues are probably the labile ligandsthat can exchange with each other in the ferric state, whereas H150 isthe tightly associated proximal ligand that always remains bound to theheme regardless of different redox or binding events (FIG. 6A and FIG.6B). EPR spectra show a small amount of histidine-ligated,pentacoordinate, high-spin heme, a possible intermediate during theligand exchange. The presence of this high-spin species with acoordination vacancy, in equilibrium with the two differenthexacoordinate ligation states of the low-spin heme, is consistent withthe observation that cyanide can bind to the ferric heme of Z-ISO.Furthermore, the substoichiometric binding of cyanide is consistent withthe MCD calibration data showing that the pentacoordinate, high-spinspecies is likely to be less than 10-20% of the total heme. When Z-ISOis reduced to the Fe(II) form, the heme ligand set becomes solelybis-histidine, thus suggesting a redox-dependent ligand switch. It isthis reduced form that is active in vitro. The Fe(II) heme can bind NOand CO, which can be used as a diagnostic probe to experimentallyinterrogate the heme for available coordination sites needed tocoordinate an exogenous ligand (FIG. 6B).

The Z-ISO has therefore been shown to be an integral membrane isomerasethat responds to redox state in performing a key step in carotenoidbiosynthesis. Isomerization is dependent on a unique cofactor carried byZ-ISO, a heme that undergoes redox-dependent ligand switching. Thereduction of the heme iron switches coordination of the heme tobis-histidine and exposes the active site for substrate binding. In theproposed mechanistic model (FIG. 6C), binding of the Z-ISO substratedisplaces the weakly associated H266 ligand, and the π electrons of the15-15′-cis carbon-carbon double bond in the substrate serve as a Lewisbase for coordination with the ferrous heme iron of Z-ISO. There is aprecedent for coordination between a carbon-carbon double-bond moietyand a heme iron, as reported for a bacterial flavohemoglobin.Spectroscopic evidence provides support for coordinate bonding betweenthe iron of the histidine-coordinated heme and a carbon-carbon doublebond of an unsaturated lipid. Binding to a transition metal such as ironcan reduce the bond order of an alkene because the π electrons aredelocalized into an empty orbital on the metal. As a result of directcoordination between the ferrous heme iron of Z-ISO and the targetdouble bond in the substrate, the single a bond remaining in thesubstrate would be free to rotate to the energetically more favorabletrans configuration, thus converting 9,15,9′-cis-ζ-carotene to9,9′-cis-ζ-carotene. As a consequence of cis-trans isomerization, theentire structure of the 40-carbon ζ-carotene substrate would change froma bulky W shape to a streamlined linear shape (FIG. 1A). These cis andtrans geometrical isomers would interact uniquely with themicroenvironment of the Z-ISO protein structure and contributedistinctly to membrane-lipid fluidity. Therefore, the altered carotenoidstructure is predicted drive release of the product from Z-ISO, thusallowing further enzymatic conversions of the Z-ISO product bydownstream enzymes. Notably, according to hard-soft acid-base theory,Fe(II), in comparison to Fe(III), is a soft Lewis acid and therebyprefers ligation to soft Lewis bases. Given that the Z-ISO substrate isa soft Lewis base, the ferrous state of Z-ISO is anticipated to presentsuperior binding kinetics and reactivity compared to the ferric state.In addition, coordination of the carotenoid double bond to the Fe(II)ion of the reduced Z-ISO heme forms a stable 18-electron coordinationcomplex. In contrast, an unsaturated and less stable (17-electron)coordination complex is generated if the substrate were coordinatingelectrons with the Fe(III) ion present in the as-purified Z-ISO heme.Thus, other than the aforementioned redox-dependent conformationalchanges, the hard-soft acid-base analysis and the 18-electron rule inorganometallic chemistry further explain the molecular basis for redoxcontrol of the isomerization activity of Z-ISO.

Heme-dependent carbon-carbon double-bond isomerization is rarelyreported in the literature. The only other double-bond isomerase knownto use heme as a cofactor is a bacterial cis-trans fatty-acid isomerase(CTI). CTI is a periplasmic enzyme that uses a c-type heme to perform asimilar cis-trans isomerization of a double bond. However, little isknown regarding the electronic structure or ligand-coordination state ofthe heme iron in this enzyme. The hypothesized catalytic mechanism ofCTI is distinct from that of Z-ISO. It has been proposed that CTIfunctions in the oxidized ferric state and that the isomerizationreaction is triggered by single-electron transfer from the double bondto the heme iron, thus oxidizing the double bond to a single bond.

The data show that reduction of the Z-ISO heme iron from Fe(III) toFe(II) is necessary for enzyme activity. The heme reduction causes aligand switch to bis-histidine and possibly triggers additionalconformational changes at the active site of Z-ISO to allow substratebinding. In the resting ferric state, Z-ISO is postulated to be in aclosed conformation excluding the binding of the bulky substrate (FIG.1A). Such redox-dependent ligand-switch phenomena have been observed inmany other hemoproteins, and the purpose of the ligand-switch behavioris to induce conformational changes that drive functional activation.This strategy appears to be a common natural approach to control thefunctional activity of hemoproteins through redox changes. For example,cytochrome cd₁, nitrite reductase must be reduced to becomecatalytically active through a mechanism that involves a redox-mediatedheme-iron ligand switch. Upon reduction, a tyrosine ligand of the d₁,heme in that enzyme is displaced to generate a coordinate vacancy forsubstrate binding. Similarly, the CO gas-sensing transcription factorCooA contains a heme cofac-tor that undergoes a ligand switch to makeCooA competent for DNA binding. Like Z-ISO, CooA goes through aredox-mediated ligand switch upon reduction of the heme iron: a cysteineaxial ligand is replaced by a histidine, thus enabling the binding of COto the heme iron at the ferrous state via displacement of the relativelyweakly bound histidine ligand. Conformational changes then follow todrive DNA binding. Another example is bacterial di-heme cyto-chrome cperoxidase (bCcP). In the resting di-ferric state of bCcPs, one heme hasa bis-histidin e axial ligand set, and the other heme has ahistidine-methionine axial ligand set. The two hemes are over 14 Åapart. A reductive activation process is generally needed for the properfunction of bCcPs: single-electron reduction of the high-potentialhistidine-methionine heme triggers a series of conformational changesthat remotely displace one of the histidine ligands of the other heme,thus allowing the access of the cosubstrate, H₂O₂, to that site.Notably, a common feature of these examples is that reduction of theinactive ferric form generates the active ferrous form, and the ligandswitch, as well as associated conformational changes, enables thebinding of substrate via the creation of a coordinate vacancy, a weaklyassociated ligand or a binding cavity. This strategy can effectivelyprotect the heme cofactor from nonproductive binding events and therebyavoids undesired side reactions.

Because the activity of Z-ISO is controlled by redox state, plastidphysiology and stress affect Z-ISO and downstream flux through thecarotenoid pathway are impacted. Plastids undergo dramatic shifts inredox status as a result of photosynthetic activity in the light andnonphotosynthetic activity in the dark. Changes in redox status areknown to be reflected through dynamic control of metabolism. Forexample, redox modulators (such as ferrodoxins and thioredoxins) adjustheme- and chlorophyll-biosynthetic activity in response to varying redoxstate. It has been proposed that carotenoid biosynthesis is also underredox control, although most of the molecular details are unknown.Mutations that inhibit expression of Z-ISO are already known to blockproduction of carotenoid-pathway end products. On the basis of theresults presented in this disclosure, changes in plastid redox state arepredicted directly influence Z-ISO activity and consequentially alterflux in the carotenoid-biosynthetic pathway. Redox tuning of Z-ISOactivity could position Z-ISO as a gatekeeper for dynamic control ofcarotenogenesis on short time scales. That is, carotenoid pools could berapidly adjusted by redox tuning of Z-ISO to respond to variable needsfor photosynthesis and signaling pathways related to stress anddevelopment.

Stress is a known factor affecting biosynthesis and action ofcarotenoids and their derivatives. NO is known to be produced directlyat the site of carotenoid biosynthesis in plant plastids in response tostress and has been shown to inhibit carotenoid accumulation. Inaddition, NO is known to inhibit heme enzymes through binding to theheme iron, especially the ferrous form. The ability of Z-ISO to bind NO,tested in the laboratory as a diagnostic heme-ligand probe, suggeststhat Z-ISO could be regulated by NO in vivo.

Hemoproteins possess a wide range of biological functions, acting asenzymes, electron transporters, gas sensors, gas transporters andtranscription factors, but double-bond isomerization is not generallyconsidered to be a prototype activity for hemoproteins. Z-ISO is theonly known heme-dependent isomerase that uses a ferrous iron, undergoesredox-mediated ligand switching and performs isomerization of a longhydrocarbon in a membrane environment. Therefore, studies of Z-ISO aspresented here open the path for further discovery and understanding ofa new class of hemoenzymes that perform double-bond isomerization inhydro-phobic environments. In the case of Z-ISO, isomerization iscritical for mediating metabolic flux of a vital plant pathway that isalso important for nutrition of humans and other animals. Furtherunderstanding of Z-ISO function will provide opportunities to bettercontrol carotenoid biosynthesis and facilitate breeding ofmore-resilient plants in a changing climate and production ofmore-nutritious crops.

Methods

General gene cloning. All gene constructs were verified by DNAsequencing.

Z-ISO expression and purification. Cloning. The maize Z-ISO codingsequence with transit sequence was commercially synthesized (Genscripl)to be codon optimized for E. coli, and restriction sites were added forcloning into SacI and BamHI sites of pUCS7. The final construct wasnamed ZmZISO ACA-less (no. 516). From this clone, the sequence encodingZ-ISO beginning at residue 49 was PCR amplified with primerstacttccaatccaatgccatgCGTCCGGCGCGTGCGGTGG (forward, SEQ ID NO. 1) andTTATCCACTTCCAATGCTACCAGGGAAGTTGGTAGCTG (reverse, SEQ ID NO. 2) andinserted by ligation-independent cloning into pMCSG9-His₁₀ (no. 646).Primer sequences in lowercase letters were for ligation-independentcloning, and those in uppercase were gene specific. The resultingconstruct, pMCSG9 Z-ISO E2 (no. 582), encodes a MBP::Z-ISO fusionprotein consisting of a decahistidine (His₁₀)-tagged MBP, at the Nterminus, which is separated from the C-terminal Z-ISO by a TEVprotease-cleavage site. The pMCSG9-His₁₀ vector was produced bymodification of vector pMCSG9 to have a His₁₀ tag instead of ahexahistidine tag and was obtained from the materials repository of theProtein Structure Initiative.

Expression and purification of the MBP::Z-ISO fusion protein. E. coliC43(DE3) overnight cultures containing pMCSG9 Z-ISO E2 (no. 582) wereused to inoculate 2×YT medium (1% yeast extract, 1.6% tryptone and 0.5%NaCl) at 1:1 00 dilution. Cultures were incubated with shaking at 200r.p.m. at 37° C. until an OD of 0.6 was reached (typically about 2 h).Protein expression was induced with 1 mMisopropyl-1-thio-D-galactopyranoside (IPTG, Gold Biotechnology) andfurther incubated for 16 h at 28° C. Cultures were centrifuged at 2,600g for IS min at 4° C., and pellets were frozen until use. Pellets wereresuspended (at a ratio of 50 ml per 8 g of cell pellets; about 40 mlper liter of initial culture) in resuspension buffer (50 mM Tris, pH7.6, Sigma-Aldrich, 300 mM NaCl and 5% glycerol) containing 0.5 mMdithiothreitol (DTT, VWR), 4 μl per 25 ml benzonase (Sigma-Aldrich), 60mg per 50 ml 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride(AEBSF, Bio-Research Products) and 0.15 mg per ml lysozyme(Sigma-Aldrich) before sonication on ice (five times, 30 s each, at 60%power) with a Vibra Cell VC600 sonicator equipped with a 3-mm taperedmicrotip (Sonics & Materials). To remove unbroken cells, thepreparations were centrifuged at about 15,000 g (11,000 r.p.m. in a type45 Ti rotor) for 15 min at 4° C. To recover the membrane fraction, thesupernatants were next centrifuged at about 120,000 g (32,000 r.p.m. ina type 45 Ti rotor) for 1 h at 4° C. The pellets containing cellmembranes were resuspended in resuspension buffer at a ratio of 8 ml perliter of initial cell culture; this was followed by sonication asdescribed above. After sonication, the volumes were increased for atotal of 40 ml per liter of starting culture. n-dodecyl β-D-maltoside(DDM, Anatrace) was added as powder to a final concentration of 1.5%.Samples were rotated end over end at 4° C. for 15 min. Cleared lysateswere incubated overnight with Ni-NTA-containing resin (Qiagen) at aratio of 300 μl resin per 40 ml of lysate for immobilized metal affinitychromatography (IMAC) in a 5-ml polypropylene column (Qiagen). Thecolumn was washed with five resin volumes of ATP wash buffer (40 mMTris, pH 7.6, 200 mM NaCl, 5% glycerol and 5 mM MgCl₂, with freshlyadded 5 mM ATP (Fisher Scientific), 0.1 mM DTT and 0.05% DDM (finalconcentrations) for 30 min, with the column under gentle rotation. Asecond wash (five resin volumes) with wash buffer (40 mM Iris, pH 7.6,400 mM NaCl and 5% glycerol) containing 0.1 mM DTT, 0.05% DDM and 30 mMhistidine (Sigma-Aldrich) was performed for 5 min (with the column undergentle rotation). The MBP::Z-ISO fusion protein was eluted with elutionbuffer (25 mM Tris, pH 7.6, 200 mM NaCl, 200 mM histidine and 5%glycerol) containing 0.1 mM DTT and 0.05% DDM at a ratio of 1 ml elutionbuffer per liter of initial culture. The protein sample was thendialyzed overnight with a Slide-A-Lyzer Dialysis cassette G2 20 Kmembrane (Thermo Scientific) against a 1,000-fold volume of buffercontaining 20 mM NaCl, 20 mM Tris, pH 7.6, 5% glycerol, 0.02% DDM and0.1 mM DTT, at 4° C. For metal analysis, 1 mM EDTA was included in thedialysis buffer, and dialysis was done for 3 h, three times. Whenneeded, the sample was concentrated with microconcentrators (MicroconJOO K, Amicon). For in vitro assays, protein was stored at −20° C. inbuffer containing 20 mM NaCl, 20 mM Tris, pH 7.6, 40% glycerol, 0.02%DDM and 0.1 mM DTT. The yield of fusion protein was about 1 mg/literculture at about 90% purity.

Expression and purification of NnrU. NnrU from Agrobacterium tumefaciensCS8 was cloned in pNYCOMPS as a C-terminal fusion to a TEVprotease-cleavage site and a His₁₀ tag (NnrU Cl, no. 744), expressed inE. coli and purified as described above for Z-ISO.

Z-ISO in vitro enzyme assay. Preparation of substrate-containingliposomes. To produce the substrate, 9,15,9′-tri-cis-ζ-carotene (tri)from E. coli BL21(DE3) cultures, 400 ml of Luria-Bertani medium (I %tryptone, 0.5% yeast extract and 1% NaCl) containing chloramphenicol (34μg per ml (Sigma-Aldrich)) was inoculated with 8 ml of overnight culturecontaining pACCRT-EBP (no. 150). Cultures were grown in the dark at 37°C., with shaking at 160 r.p.m. for 8 h before induction with 10 mM IPTG.Cultures were further incubated at 28° C., with shaking at 100 r.p.m.for 40 h and for an additional 2 d without shaking. Cells werecentrifuged at 2,600 g, and pellets were resuspended in a total of 40 mlof methanol, distributed in four conical tubes with equal volumes ofextract and then sonicated twice on ice (30 s each, at 60% power) with aVibra Cell VC600 sonicator equipped with a tapered 3-mm microtip (Sonics& Materials). Extracts were centrifuged at 2,600 g for 10 min, andsupernatants were transferred to 15-ml conical tubes and evaporatedunder nitrogen gas in the dark. Dried samples were resolubilized in 300μl of methanol, transferred to 1.5-ml microfuge tubes, frozen at −80° C.for 1 h and centrifuged at 16,000 g at 4° C. Extractions were thencombined, and 1 ml was used to prepare liposomes. Cells also accumulate9,9′-tri-cis-ζ-carotene (di), and therefore enzymatic conversion ismeasured as the ratio of di to tri isomers. For preparation ofliposomes, 1 ml of substrate extract (58 μM, estimated by spectroscopy,with the molar extinction coefficient for ζ-carotene, ε₄₀₀, equal to138,000) was mixed with 35 μl of soybean L-α-phosphatidylcholine(Sigma-Aldrich, 99% pure) (20 mg per ml in methanol). The mixture wasdried under N₂, and this was followed by addition of 800 μl sonicationbuffer (25 mM HEPES, pH 7.8, 100 mM NaCl and 10% glycerol) andsonication on ice with a Vibra Cell VC600 sonicator equipped with a 3-mmtapered microtip (Sonics & Materials) for 1 mM, at intervals of 10 s at20% power.

In vitro reactions. To assemble a biphasic assay system (final volume of400 μl), purified, MBP::Z-ISO fusion protein (10 μM final concentration)was incubated with 15 μl of AcTEV protease (ISO units, Invitrogen) for 2min at room temperature. To generate reducing conditions, freshlyprepared sodium dithionite (Sigma-Aldrich, 85% pure) was added to afinal concentration of 10 mM in the assay. To initiate the reaction, 200μl of substrate-containing liposomes (for a final concentration of 36.5μM substrate) was added, and reactions were overlaid with N₂ gas beforecapping. Reactions were incubated at 28° C. under continuous shaking at130 r.p.m. for 3 h in the dark (to prevent photoisomerizalion). Reactions in the absence of sodium dithionite were also assembled. As anegative control, heat-denatured (10 min at 100° C.) MBP::Z-ISO fusionwas used. Reactions were extracted by addition of 1 ml of petroleumether/diethyl ether 2:1 (v/v), and the organic phase was collected,dried under N₂ dissolved in 150 μl methanol and 100 μl and separated byHPLC as described below. All reactions were replicated three times.

Bioinformatics. MEMSAT3, which has been experimentally validated anddetermined to be one of the better predictors of membrane topology, wasused to predict transmembrane domains in maize Z-ISO. Thetransit-peptide sequence was predicted with the ChloroP program aspreviously reported. The Z-ISO protein sequence from Zea mays wasanalyzed by the fold-recognition program, LOOPP (LOOPP parallel driverv7.0 with LOOPP v3.20) which modeled 276 residues of Z-ISO onto adi-iron protein, PDB 2INP).

HPLC analysis. HPLC separations were performed on a Waters HPLC systemequipped with a 2695 separation module, 996 photodiode array detector(Waters) and Empower I software (Waters). A C30 Develosil 5 u RPAQUEO US(250×4.6 mm) column from Phenomenex (Nomura Chemical) was used. Forisocratic separation of 100 μl of carotenoid extract, a mobile phase offour parts water, 66 parts methanol (VWR, HPLC grade) and 30 partsmethyl-t-butyl-ether (VWR, HPLC grade), at a constant flow rate of 1 mlper min for 80 min, was applied. Identification of ζ-carotene isomerswas based on elution time and spectra, as previously published.

Z-ISO localization. Transient expression of Z-ISO in protoplasts. A fullcopy of maize Z-ISO without a stop codon was amplified from pColZmZ-ISOIplasmid (no. 497), with forward primer 2793(5′-atctctagaATGGCCTCCCAGCTCCGCCTCCACC, SEQ ID NO. 3), containing anXbal site, and reverse primer 2794 (5′-atcggatccCCAGGGAAGTTGGTAGCTGGATGC, SEQ ID NO. 4), containing a BamHI site, andwas inserted into the pUC35S-sGFP-Nos vector (digested with Xbal andBamHI), to produce the pUC35S-M-ZISO-sGFP-Nos plasmid (no. 568), whichwas used for transient expression. Transient expression of Z-ISO-GFP inmaize green leaf protoplasts was performed.

In vitro import of Z-ISO into chloroplasts. A full copy of the maizeZ-ISO gene, without a stop codon, was amplified from pColZmZ-ISOl (no.497) with forward primer 2851 (ccacctgcaGAATTCtatggcctc, SEQ ID NO. 5),containing an EcoRI site, and reverse primer 2854(gtcTCTAGAttatttttcaaattgaggatgagaccaccagggaagttggtagct, SEQ ID NO. 6)),containing a streptavid in tag and XbaI site, and was inserted intovector pTnT (Promega), which was digested with the same restrictionenzymes to yield plasmid pTnT-M-ZISO-Strep (no. 570). pTnT-M-ZISO-Strepwas used as a template for in vitro protein synthesis. In vitro proteinsynthesis and import of Z-ISO into isolated pea chloroplasts wereperformed as previously described. After import, chloroplasts weretreated with thermolysin (+) to remove nonspecifically bound protein.Chloroplasts were also fractionated into soluble (S) and membrane (M)fractions, including envelope and thylakoid; an equal amount of themembrane fraction as in M was alkaline treated (MA) to remove peripheralmembrane proteins, thus indicating that Z-ISO is a membrane integralprotein. Alkaline treatment has been shown to remove loosely associatedperipheral membrane proteins rather than integral membrane proteins,which remain membrane associated.

Identification of a Z-TSO complex. After [³⁵S] methionine-labeled Z-ISOwas imported into chloroplasts, the chloroplast sample was treated with0.5% Triton X-100 to isolate protein complexes under native conditions.The sample was then separated into individual complexes by native gelelectrophoresis in a NativePAGE Novex 4-16% gel (Invitrogen, LifeTechnologies), according to the instructions of the manufacturer. Thegel was then dried and the radioactive band detected by a Phosphorimagersystem (Amersham, GE Life Sciences). The size of the band was estimatedin comparison to NativeMark protein marker (Invitrogen, LifeTechnologies).

Detection of metals in Z-ISO. Inductively coupled plasma opticalemission spectrometry (ICP-OES). Samples of MBP::Z-ISO (greater than 90%pure) were dialyzed three limes each against a 1,000-fold volume ofbuffer (20 mM Tris, pH 7.6, 20 mM NaCl, 5% glycerol, 0.02% DDM, 0.1 mMDTT and 1 mM EDTA) and injected into a Spectro Genesis inductivelycoupled optical emission spectrometer to measure the concentrations ofiron at 238, 204 nm and 259, 941 nm and sulfur at 180, 731 nm For 23 μMprotein, 15.4 μM iron was detected. Levels of calcium, copper, nickel,magnesium, manganese, molybdenum or zinc were insignificant.

Detection of heme. Pyridine hemochrome assay. To determine whether thechromophore bound to Z-ISO was heme, a pyridine hemocrome assay wasperformed. Purified protein (750 μl) was mixed with 75 μI of 1 N NaOH(Fisher Scientific), 175 μl of pyridine (Sigma-Aldrich) and 2 mg ofsodium dithionite. The UV-vis absorption spectrum was immediatelyrecorded and compared with the spectrum of the initial purified samplebefore addition of dithionite. The presence of the Soret band at 414 nmin the ferric stale and the presence of the Sorel band (418 nm) andappearance of the α-β bands at 555 and 530 nm, respectively, in theferrous state were used as evidence for the presence of heme.

Heme stain. Heme staining, based on heme peroxidase activity, wasperformed. Protein samples were separated on a NuPAGE Bis-Tris 12%polyacrylamide gel (Invitrogen). The gel was rinsed with water for 15 sand then incubated for 1 h in the dark in a solution containing 30 ml of40 mM TMBZ (3,3,5,5′-tetramethylbenzidine, Sigma-Aldrich) in methanol;this was followed by the addition of 70 ml of 0.25 M sodium acetate, pH5.0 (Sigma-Aldrich). Then 5 ml of 3% hydrogen peroxide was added, andsamples were mixed well until a signal corresponding to the MBP Z-ISOband appeared. The gel background was removed by destaining 15 min with3:7 isopropanol/0.25 M sodium acetate.

Binding of CN⁻. MBP::Z-ISO, 75.46 KDa (1.58 mg per ml, 21 μM), purifiedas described above, was incubated with KCN (Sigma-Aldrich, greater thanor equal to 96% pure) at a final concentration of 2 mM. The UV-visspectrum was recorded before and immediately after addition and mixingof KCN. The experiment was repeated except that MBP::Z-ISO was firstreduced with sodium dithionite (2 mg, added as dry powder) beforeaddition of KCN.

UV-visible spectroscopy Z-ISO difference spectra. In the reduced minusoxidized spectrum, the graph was obtained by subtraction of the UV-visspectrum of the dithionite-reduced enzyme from the spectrum of theenzyme as purified. In the CO difference spectrum, the graph wasobtained by subtraction of the UV-vis spectrum of the dithionite-reducedenzyme from the spectrum of the enzyme that was dithionile reduced andthen treated with CO. For the cyanide difference spectra, the graph wasobtained by subtraction of the UV-vis spectrum of the as-purified enzymefrom the spectrum of the as-purified enzyme that was treated withcyanide or by subtraction of the UV-vis spectrum of thedithionite-reduced enzyme from the spectrum of the dithionite-reducedenzyme that was treated with cyanide.

Electron spin resonance spectroscopy. X-band EPR spectra of Z-ISO wererecorded in the perpendicular mode on a Bruker ER200D spectrometercoupled with a 4116DM resonator at 100-kHz modulation frequency. Themeasurement temperature was maintained at 10 K with an ESR910liquid-helium cryostat and an ITC503 temperature controller from OxfordInstruments. The reduced Z-ISO protein was generated by dithionatereduction under anaerobic conditions. Nitric oxide was anaerobicallyintroduced through a gas-tight syringe to the headspace of the quartzEPR tubes containing reduced Z-ISO. An argon flush was maintained abovesamples to protect them from oxidation by O₂ and to minimize ananomalous EPR signal near g of 2, which derives from NO.

Magnetic circular dichroism. MCD spectra were measured on a Jasco J815spectropolarimeter fitted with a Jasco MCD-1 B magnet at amagnetic-field strength of 1.41 T at 4° C. with a quartz cuvette with0.5-cm path length and interfaced with a Silicon Solutions PC through aJASCO IF-815-2 interface unit. MCD data acquisitions and manipulationswere carried out with JASCO software, as reported previously.

Site-directed mutagenesis and functional complementation in E. coli. Themaize Z-ISO cDNA coding sequence from pColZmZ-ISOl plasmid (no. 497) wasused as a template to PCR-amplify and subclone Z-ISO lacking thetransit-peptide sequence (amino acids 1-46). For PCR, forward primer(5′-cgggatcct CACGCTCGTCCCGCCCGTGCG-3′, SEQ ID NO. 7) containing a BamHIsite and reverse primer (5′-gcgtcgaccTACCAGGGAAGTTGGTAGCT-3′, SEQ ID NO.8) containing a SalI site were used. Lowercase letters in primerscontain restriction sites, and uppercase letters contain gene-specificsequences. The resulting PCR product was further inserted into the BamHIand SalI sites of pCOLADuet-1, forming a histidine-tag::Z-ISO fusion,and the vector was named pCola Zm Z-ISO NTP (no. 579). pCola Z-ISO NTPwas then used as template to perform substitutions of conserved residuesto alanine. Residue substitutions used in this study were H150 (no. 797,pCol Zm Z-ISO NTP H150A), H266 (no. 798, pCol Zm Z-ISO NTP H266A) andC263 (no. 796, pCol Zm Z-ISO NTP C263A). Other residue substitutionstested were made in the pColZmZ-ISOI plasmid (no. 497): H191 (no. 523,pCol Zm Z-ISO H 191A), H208A (no. 528, pCol Zm Z-ISO H208A), H241 (no.529, pCol Zm Z-!SO H241A), H253 (no. 530, pCol Zm Z-ISO H253A). H354(no. 532, pCol Zm H354A), H285 (no. 525, pCol Zm H285A) and H286A (no.526, pCol Zm Z-ISO H286A). For H150, H266 and C263, mutations were alsocreated in the MBP::Z-ISO fusion construct with the pMCSG9 Z-ISO E2plasmid (no. 582) as a template to generate the MBP::Z-ISO mutantversions pMCSG9 Z-ISO E2 H150A (no. 619), pMCSG9 Z-ISO E2 H266A (no.620) and pMCSG9 Z-ISO E2 C263A (no. 801), which were expressed in E.coli as described above. Reactions for mutagenesis were performed withthe QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) andprimers designed to incorporate the desired substitution. For functionaltesting, the Z-ISO mutant genes were further transformed into E. colicells containing the plasmid pACCRT-EBP (no. 150), which confersaccumulation of ζ-carotene. For functional complementation, mutant Z-ISOgenes were introduced into E. coli cells accumulating the Z-ISOsubstrate. Carotenoids were extracted from the bacteria containing thevarious enzyme variants and were subjected to HPLC analysis to quantifythe ratio of product (9,9′-di-cis-ζ-carotene) to substrate (9,15,9′-tri-cis-ζ-carotene). Cells with empty vector also accumulate asmall amount of product. Therefore, enzyme activity is judged by theincrease over this background level. Specifically, 1-ml volumes ofsaturated cultures in Luria-Bertani medium (1% tryptone, 0.5% yeastextract and 1% NaCl) were added to 50 ml of fresh medium and then grownin the dark at 37° C. at 200 r.p.m. for 8 h before induction with 10 mMIPTG and further incubation for 40 h at 28° C. with slow shaking (100r.p.m.) and an additional 2 d without shaking. For carotenoidextraction, bacterial cultures were centrifuged at 2,600 g for 10 min.Pellets were resuspended in 5 ml of methanol containing 1% of butylatedhydroxytoluene (Sigma-Aldrich, greater than or equal to 99% pure) andsonicated with a Vibra Cell VC600 sonicator equipped with a 3-mm taperedmicrotip (Sonics & Materials) on ice twice (30 s each, at 60% power).Extracts were centrifuged at 2,600 g for 10 min, supernatants weretransferred to 15-ml conical tubes, and extracts were evaporated undernitro-gen gas in the dark. Dried samples were resolubilized in 500 μl ofmethanol, transferred to 1.5-ml tubes, frozen at −80° C. for 1 h andcentrifuged at 16,000 g at 4° C., and supernatants were used for HPLCseparation as described above. Complementation experiments werereplicated three times.

Immunodetection of Z-ISO. For antibody generation, 2 mg of MBP::Z-ISOprotein (no. 582) was digested with TEV protease to generate free Z-ISO.Samples were separated with the NuPAGE system from Invitrogen. Proteinbands corresponding to Z-ISO were excised and shipped to LampireBiological Laboratories for rabbit immunization. Polyclonal antibodiesagainst Z-ISO were generated in two rabbits identified as no. 190202 andno. 190203. For immunodetection, protein samples were separated byelectrophoresis with the NuPAGE system (Invitrogen). Reducing conditionsin the samples were generated with DTT (100 mM). Proteins weretransferred onto nitrocellulose membranes (Optitran, Whatman) with anelectrophoretic transfer cell (Criterion Blotter, Bio-Rad) at 20 Vovernight, ° C. with Ix transfer buffer (25 mM Tris, 192 mM glycine and20% (v/v) methanol). The membranes were then incubated in blockingbuffer (1×PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mMKH₂PO₄), 3% bovine serum albumin (Fisher Scientific) and 1% Tween-20(Sigma-Aldrich)) for 1 h at room temperature, then for 1 h at roomtemperature with anti-Z-150 polyclona 1 antibody (1:2,000) produced inrabbit no. 190203. After washing, the membranes were incubated withhorseradish peroxidase-conjugated goat anti-rabbit IgG (Invitrogen) for1 h at RT and washed with 1×PBS buffer containing 1% Tween-20 for 15min; this was followed by four additional washes of 5 min each.Immunoreactions were visualized with the Super Signal West Dura kit(Thermo Scientific). Fluorescence signals were captured with a G:box(ChemiXT4) from Syngene with Genesys V1.3.1.0 software.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal language of the claims.

What is claimed is:
 1. A method for isomerizing a double bond, themethod comprising: exposing a substrate to an isomerase enzyme, whereinthe isomerase enzyme comprises a redox-regulated ligand switch and hemeb cofactor, wherein the heme b cofactor comprises an iron having an iron(III) oxidation state and the substrate comprises a double bond in thesubstrate with a cis stereochemistry; exposing the isomerase enzyme to areducing agent such that the isomerase enzyme changes from the iron(III) oxidation state to an iron (II) oxidation state, wherein theisomerase enzyme isomerizes a double bond when in the iron (II)oxidation state and does not isomerize the double bond when in the iron(III) oxidation state; and permitting the double bond in the substrateto undergo isomerization from the cis stereochemistry to a transstereochemistry, wherein the isomerization is catalyzed by the isomeraseenzyme.
 2. The method as recited in claim 1, wherein the double bond inthe substrate is a carbon-carbon double bond.
 3. The method as recitedin claim 1, wherein the double bond in the substrate is anitrogen-oxygen double bond.
 4. The method as recited in claim 1,wherein the isomerase enzyme is at least 70% homologous to either SEQ IDNO. 9 or SEQ. ID NO.
 10. 5. The method as recited in claim 1, whereinthe isomerase enzyme is at least 80% homologous to either SEQ ID NO. 9or SEQ. ID NO.
 10. 6. The method as recited in claim 1, wherein theisomerase enzyme is at least 90% homologous to either SEQ ID NO. 9 orSEQ. ID NO.
 10. 7. The method as recited in claim 1, wherein theisomerase enzyme is at least 95% homologous to either SEQ ID NO. 9 orSEQ. ID NO.
 10. 8. The method as recited in claim 1, wherein theisomerase enzyme is at least 95% homologous to SEQ ID NO.
 9. 9. Themethod as recited in claim 1, wherein the isomerase enzyme is at least95% homologous to SEQ ID NO.
 10. 10. The method as recited in claim 1,wherein the isomerase enzyme has fewer than four hundred residues andcomprises SEQ ID NO.
 11. 11. The method as recited in claim 1, whereinthe exposing the isomerase enzyme to the reducing agent occurs in vitro.12. The method as recited in claim 1, wherein the substrate is acarotenoid.
 13. A method for isomerizing a double bond, the methodcomprising sequential steps of: exposing a carotenoid substrate to anisomerase enzyme, wherein the isomerase enzyme comprises aredox-regulated ligand switch and heme b cofactor, wherein the heme bcofactor comprises an iron having an iron (III) oxidation state and thecarotenoid substrate comprises a double bond in the carotenoid substratewith a cis stereochemistry, wherein the isomerase enzyme has fewer thanfour hundred residues and comprises SEQ ID NO. 11; exposing theisomerase enzyme to a reducing agent in vitro such that the isomeraseenzyme changes from the iron (III) oxidation state to an iron (II)oxidation state, wherein the isomerase enzyme isomerizes a double bondwhen in the iron (II) oxidation state and does not isomerize the doublebond when in the iron (III) oxidation state; and permitting a doublebond in the carotenoid substrate to undergo isomerization from the cisstereochemistry to a trans stereochemistry, wherein the isomerization iscatalyzed by the isomerase enzyme.
 14. The method as recited in claim13, wherein the isomerase enzyme is at least 70% homologous to eitherSEQ ID NO. 9 or SEQ. ID NO.
 10. 15. The method as recited in claim 13,wherein the isomerase enzyme is at least 80% homologous to either SEQ IDNO. 9 or SEQ. ID NO.
 10. 16. The method as recited in claim 13, whereinthe isomerase enzyme is at least 90% homologous to either SEQ ID NO. 9or SEQ. ID NO.
 10. 17. The method as recited in claim 13, wherein theisomerase enzyme is at least 95% homologous to either SEQ ID NO. 9 orSEQ. ID NO. 10.